The present invention provides methods, host cells, and vectors which permit efficient production of recombinant parvovirus virions. In particular, the present invention relates to parvovirus helper functions that provide for high-efficiency recombinant parvovirus production but reduce the potential of generating replication competent particles.
Parvoviruses vectors, such as adeno-associated virus (AAV) vectors are useful for gene therapy. In general, recombinant adeno-associated virus (rAAV) vectors are generated by transfection of an AAV vector plasmid and a helper plasmid in the presence of helper virus infection (Samulski, et al. (1989) J Virol 63: 3822-3828). The AAV vector is constructed by replacing the whole coding region of the AAV genome with a transgene. This creates a defective AAV vector which is incapable of replication. In order to provide the necessary helper functions, a helper plasmid can be constructed. The helper plasmid contains the AAV Cap and/or Rep coding region, but lacks the AAV inverted terminal repeat sequences. Accordingly, the helper plasmid can neither replicate nor package itself. After the AAV helper plasmid and the AAV vector are introduced into a host cell, the transfected cells can be infected with a helper virus, for example, an adenovirus, which, among other functions, transactivates the AAV promoters present on the helper plasmid that direct the transcription and translation of AAV Rep and Cap regions. Upon subsequent culture of the host cells, recombinant AAV virions (harboring the transgene) are produced.
Although there is no overlapping sequence between the AAV vector and the helper plasmid, the probability of generating replication competent AAV (rcAAV) particles through non-homologous recombination, is relatively high (Allen et al. (1997) J Virol 71: 6816-6822). These replication competent particles affect transgene expression (Grimm, et al. (1999) Hum Gene Ther 10: 2445-2450), are a safety hazard in applications of AAV vectors for human gene therapy, and also reduce the yield of recombinant AAV virions.
Previous attempts to address the problem of rcAAV particles includes using heterologous promoters for driving the Rep coding and Cap region, separating the Cap and Rep coding regions into different vectors (See Allen, et al. (1997) J Virol 71: 6816-6822 and Flotte, et al. (1995) Gene Ther 2: 29-37), and using truncated AAV terminal repeat sequences (Wang, et al. (1998) J Virol 72: 5472-5480). Although these approaches reduced the number of replication competent particles, the replication competent particles were still present in large scale preparations. Accordingly, a need exists for methods and compositions of producing recombinant viral vectors without the presence for contaminating replication competent particles. A need also exists for methods of producing recombinant AAV virions without the presence of contaminating replication competent particles.